U.S. patent application number 15/722877 was filed with the patent office on 2018-02-01 for systems and methods for monitoring the amplification of dna.
This patent application is currently assigned to Canon U.S. Life Sciences, Inc.. The applicant listed for this patent is Canon U.S. Life Sciences, Inc.. Invention is credited to Kenton C. Hasson.
Application Number | 20180030513 15/722877 |
Document ID | / |
Family ID | 41431637 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180030513 |
Kind Code |
A1 |
Hasson; Kenton C. |
February 1, 2018 |
SYSTEMS AND METHODS FOR MONITORING THE AMPLIFICATION OF DNA
Abstract
A system and method for amplifying and detecting nucleic acids
are disclosed. In one embodiment, the system includes: a
microfluidic device comprising a channel for receiving a sample of
solution containing real-time PCR reagents; a temperature control
system configured to cycle the temperature of the sample; an
excitation source for illuminating the sample; a fiber optic probe
comprising (i) an optical fiber having a distal end and a proximal
end and (ii) a probe head connected to the distal end of the
optical fiber and positioned between the distal end of the optical
fiber and the channel; and a detector configured to detect
emissions exiting the proximal end of the optical fiber.
Inventors: |
Hasson; Kenton C.;
(Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Canon U.S. Life Sciences, Inc. |
Rockville |
MD |
US |
|
|
Assignee: |
Canon U.S. Life Sciences,
Inc.
Rockville
MD
|
Family ID: |
41431637 |
Appl. No.: |
15/722877 |
Filed: |
October 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14697036 |
Apr 27, 2015 |
9777318 |
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15722877 |
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12144223 |
Jun 23, 2008 |
9017946 |
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14697036 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/686 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A system for performing real-time PCR, comprising: a sample
container for containing a sample of solution containing real-time
PCR reagents; a temperature control system configured to cycle the
temperature of the sample; an excitation source for illuminating
the sample; a fiber optic probe comprising: a bundle of optical
fibers including a central optical fiber surrounded by a plurality
of outer optical fibers, and a probe head connected to a distal end
of the central optical fiber and positioned between the distal end
of the central optical fiber and the sample container for
containing the sample, and a detector configured to detect
emissions exiting the proximal end of the central optical fiber,
wherein the excitation source is optically connected to each of
said plurality of outer optical fibers such that when the
excitation source emits excitation light, the excitation light
enters the outer optical fibers and then exits the outer optical
fibers through a distal end of the optical fibers.
2. The system of claim 1, wherein the excitation source comprises
at least two light emitting devices, wherein each of the at least
two light emitting devices is optically connected to at lest one of
the outer optical fibers.
3. The system of claim 1, wherein the sample container is in the
form of a channel.
4. The system of claim 1, wherein the probe head comprises a ball
lens or a gradient index lens.
5. The system of claim 1, wherein the diameter of the probe head is
less than about 5 millimeters.
6. The system of claim 5, wherein the diameter of the probe head is
less than about 2 millimeters.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/697,036, filed on Apr. 27, 2015, which is a divisional
of U.S. patent application Ser. No. 12/144,223, filed on Jun. 23,
2008, now U.S. Pat. No. 9,017,946, the disclosures of each of which
are hereby incorporated by reference in their entireties.
BACKGROUND
Field of the Invention
[0002] This invention pertains to systems and methods for
amplifying and detecting nucleic acids. In one embodiment, it
pertains to methods for monitoring a polymerase chain reaction
(PCR) in a microfluidic system.
Discussion of the Related Art
[0003] The detection of nucleic acids is central to medicine,
forensic science, industrial processing, crop and animal breeding,
and many other fields. The ability to detect disease conditions
(e.g., cancer), infectious organisms (e.g., HIV), genetic lineage,
genetic markers, and the like, is ubiquitous technology for disease
diagnosis and prognosis, marker assisted selection, correct
identification of crime scene features, the ability to propagate
industrial organisms and many other techniques. Determination of
the integrity of a nucleic acid of interest can be relevant to the
pathology of an infection or cancer. One of the most powerful and
basic technologies to detect small quantities of nucleic acids is
to replicate some or all of a nucleic acid sequence many times, and
then analyze the amplification products. PCR is perhaps the most
well-known of a number of different amplification techniques.
[0004] PCR is a powerful technique for amplifying short sections of
DNA. With PCR, one can quickly produce millions of copies of DNA
starting from a single template DNA molecule. PCR includes a three
phase temperature cycle of denaturation of DNA into single strands,
annealing of primers to the denatured strands, and extension of the
primers by a thermostable DNA polymerase enzyme. This cycle is
repeated so that there are enough copies to be detected and
analyzed. In principle, each cycle of PCR could double the number
of copies. In practice, the multiplication achieved after each
cycle is always less than 2. Furthermore, as PCR cycling continues,
the buildup of amplified DNA products eventually ceases as the
concentrations of required reactants diminish. For general details
concerning PCR, see Sambrook and Russell, Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a
joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc., (supplemented through 2005) and PCR
Protocols A Guide to Methods and Applications, M. A. Innis et al.,
eds., Academic Press Inc. San Diego, Calif. (1990).
[0005] Real-time PCR refers to a growing set of techniques in which
one measures the buildup of amplified DNA products as the reaction
progresses, typically once per PCR cycle. Monitoring the
accumulation of products over time allows one to determine the
efficiency of the reaction, as well as to estimate the initial
concentration of DNA template molecules. For general details
concerning real-time PCR see Real-Time PCR: An Essential Guide, K.
Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
[0006] Several different real-time detection chemistries now exist
to indicate the presence of amplified DNA. Most of these depend
upon fluorescence indicators that change properties as a result of
the PCR process. Among these detection chemistries are DNA binding
dyes (such as SYBR.RTM. Green) that increase fluorescence
efficiency upon binding to double stranded DNA. Other real-time
detection chemistries utilize Foerster resonance energy transfer
(FRET), a phenomenon by which the fluorescence efficiency of a dye
is strongly dependent on its proximity to another light absorbing
moiety or quencher. These dyes and quenchers are typically attached
to a DNA sequence-specific probe or primer. Among the FRET-based
detection chemistries are hydrolysis probes and conformation
probes. Hydrolysis probes (such as the TaqMan.RTM. probe) use the
polymerase enzyme to cleave a reporter dye molecule from a quencher
dye molecule attached to an oligonucleotide probe. Conformation
probes (such as molecular beacons) utilize a dye attached to an
oligonucleotide, whose fluorescence emission changes upon the
conformational change of the oligonucleotide hybridizing to the
target DNA.
[0007] A number of commercial instruments exist that perform
real-time PCR. Examples of available instruments include the
Applied Biosystems PRISM 7500, the Bio-Rad iCylcer, and the Roche
Diagnostics LightCycler 2.0. The sample containers for these
instruments are closed tubes which typically require at least a 10
.mu.l volume of sample solution. If the lowest concentrations of
template DNA detectable by a particular assay were on the order of
one molecule per microliter, the detection limit for available
instruments would be on the order of tens of targets per sample
tube.
[0008] More recently, a number of high throughput approaches to
performing PCR and other amplification reactions have been
developed, e.g., involving amplification reactions in microfluidic
devices, as well as methods for detecting and analyzing amplified
nucleic acids in or on the devices. Thermal cycling of the sample
for amplification is usually accomplished in one of two methods. In
the first method, the sample solution is loaded into the device and
the temperature is cycled in time, much like a conventional PCR
instrument. In the second method, the sample solution is pumped
continuously through spatially varying temperature zones.
[0009] U.S. patent application Ser. No. 11/505,358, entitled,
"Real-time PCR in micro-channels," which is assigned to the
assignee of this application and which is incorporated herein by
this reference in its entirety, describes, among other things, a
novel method to acquire real-time PCR data in a microfluidic
system. One of the steps in that method is to capture an image of a
fluorescent signal along the length of at least one microfluidic
channel.
[0010] A conventional apparatus to capture an image of a
fluorescent signal is illustrated in FIG. 1. As illustrated in FIG.
1, the light emitted from the material under study is collected by
a high numerical aperture objective and the light is re-imaged onto
a two-dimensional detector array.
[0011] A reason for using a high numerical aperture objective to
collect luminescence is that the solid angle subtended is higher,
and therefore the photon collection efficiency is higher, than that
achieved using a low numerical aperture objective. In certain
cases, collection efficiency may be an important parameter because,
in certain cases, emitted light flux is often so low that signal
levels at the detector are weak. Therefore, at least in certain
cases, it is desirable to maximize collection efficiency.
[0012] The drawback of using a high numerical aperture microscope
objective is that the imaged area is small. The effective field of
view of a conventional fluorescence microscope imaging system might
have a linear dimension of 1 mm or smaller. This becomes a problem
when the region of interest on a microfluidic chip is larger (e.g.,
if the length and width are in the range of 10-100 mm).
[0013] One strategy to address the problem of imaging a large
region of interest with high collection efficiency is to use an
optical system with large diameter optics. This strategy has a
benefit that most or all of the region of interest may be imaged
simultaneously. An example of this approach is illustrated in U.S.
Patent Application 2006/0006067, entitled, "Optical Lens System and
Method for Microfluidic Devices," which describes a multi-element
lens system.
[0014] Another strategy would be to translate the sample holder
with respect to the optical system or vice versa (e.g. in a raster
pattern) to collect pixel data in series. An example of this
approach is described in U.S. Pat. No. 5,631,734, entitled, "Method
and Apparatus for Detection of Fluorescently Labeled Materials."
This patent describes a system for collecting fluorescence data
from a substrate, for example a DNA microarray, in which the
substrate is held by an x-y-z translation stage and translated in
front of a microscope objective.
[0015] PCT publication WO 2005/075683 A1, entitled, "High
Throughput Device for Performing Continuous-Flow Reactions,"
describes a continuous-flow PCR device that uses a fused silica
capillary wrapped into a helix around three temperature-controlled
blocks. This publication shows a microscope objective lens being
scanned transverse to the windings. Although the description is
short on detail, presumably an entire optical imaging system,
including lenses, beam-splitters, filters, and detectors, would
have to be scanned along as well.
[0016] U.S. Pat. No. 5,928,907, entitled, "System for Real Time
Detection of Nucleic Acid Amplification Products," describes a
system for real-time PCR monitoring that uses a fiber optic and a
lens to capture fluorescence from a closed, Eppendorf-style sample
tube. The sample tube volume was 200 ul, and the fiber optic and 8
mm diameter collection lens were fixed with respect to the tube,
looking down through the top of the tube and the airspace over the
sample solution.
[0017] U.S. Patent Application 2005/0069257 A1, "Fiber Lens with
Multimode Pigtail" gives an example of a miniature lens system that
is permanently affixed to the end of an optical fiber. Further
examples of miniature fiber coupling systems can be found in
product literature by Corning Inc. for lensed fibers, tapered
fibers, and gradient index fibers and lenses. These devices are
used typically in telecommunication equipment, for example, for
coupling light from a semiconductor diode laser into an optical
fiber, or for coupling light from one fiber into another fiber.
SUMMARY OF THE INVENTION
[0018] The present invention provides, among other things, improved
systems and methods for capturing an image of a fluorescent signal.
In addition, the present invention may be useful in a variety of
additional applications.
[0019] A system according to an embodiment of the invention
includes: a microfluidic device comprising a channel for receiving
a sample of solution containing real-time PCR reagents; a
temperature control system configured to cycle the temperature of
the sample; and an imaging system for detecting emissions from the
sample, wherein the imaging system comprises: an excitation source
for illuminating the sample, a fiber optic probe comprising (i) an
optical fiber having a distal end and a proximal end and (ii) a
probe head connected to the distal end of the optical fiber and
positioned between the distal end of the optical fiber and the
channel, and a detector configured to detect emissions exiting the
proximal end of the optical fiber.
[0020] The probe head had may be positioned directly above the
channel and may be positioned no more than about 10 millimeters
from the top of the channel. The system may also include: a
positioning system configured to scan the fiber optic probe over at
least a portion of the channel and a pump for causing the sample to
flow through the channel. In such embodiments, the positioning
system may be configured to scan the fiber optic probe over the
portion of the channel at a speed that is greater than the speed at
which the sample is expected to flow through the channel.
[0021] In some embodiments, the imaging system may also include a
second fiber optic probe comprising (i) an optical fiber having a
distal end and a proximal end and (ii) a probe head connected to
the distal end of the optical fiber and positioned between the
distal end of the optical fiber and the channel. In such a system,
the first fiber optic probe may have a first field of view and the
second fiber optic probe may have a second field of view, wherein a
first portion of the channel is within the first field of view but
not the second field of view and a second portion of the channel is
within the second field of view but not the first field of
view.
[0022] With respect to the probe head, in some embodiments, the
probe head includes a ball lens, a gradient index lens, a liquid
lens, or a high index liquid. The probe head may have a diameter
between about 0.1 millimeters (mm) and 5 mm, but more preferably
between about 0.5 mm and 2 mm. Additionally, in some embodiments
the distance between the probe head and the channel is less than 10
mm (e.g., about 1 mm in some embodiments). With respect to the
optical fiber, in some embodiments, the optical fiber includes: a
multimode fiber, a liquid filled fiber, a photonic crystal
fiber.
[0023] In some embodiments, the excitation source is optically
connected to the optical fiber such that when the excitation source
emits excitation light, the excitation light enters the optical
fiber and then exits the optical fiber through the distal end of
the optical fiber.
[0024] In some embodiments, the microfluidic device further
includes a second channel for receiving a second sample of solution
containing real-time PCR reagents, and the imaging system further
comprises a second fiber optic probe comprising (i) an optical
fiber having a distal end and a proximal end and (ii) a probe head
connected to the distal end of the optical fiber and positioned
between the distal end of the optical fiber and the second channel,
and a second detector configured to detect emissions exiting the
proximal end of the optical fiber of the second fiber optic probe.
In such embodiments, the system may include a positioning system
configured to scan the first fiber optic probe over at least a
portion of the first channel and simultaneously scan the second
fiber optic probe over at least a portion of the second
channel.
[0025] In some embodiments, one or more filters may be positioned
between the proximal end of the optical fiber and the detector. The
one or more filters may include a tunable wavelength filter.
[0026] A system according to another embodiment includes: a sample
container for containing a sample of a solution containing
real-time PCR reagents; a temperature control system configured to
cycle the temperature of the sample; an excitation source for
illuminating the sample; a fiber optic probe comprising: a bundle
of optical fibers including a central optical fiber surrounded by a
plurality of outer optical fibers, and a probe head connected to a
distal end of the central optical fiber and positioned between the
distal end of the central optical fiber and the sample container,
and a detector configured to detect emissions exiting the proximal
end of the central optical fiber, wherein the excitation source is
optically connected to each of the plurality of outer optical
fibers such that when the excitation source emits excitation light,
the excitation light enters the outer optical fibers and then exits
the outer optical fibers through a distal end of the optical
fibers. The sample container may be in the form of a channel.
[0027] In some embodiments, the excitation source comprises at
least two light emitting devices, wherein each of the at least two
light emitting devices is optically connected to at lest one of the
outer optical fibers.
[0028] A method according to some embodiments of the invention
includes: (a) moving a sample of test solution containing real-time
PCR reagents through a channel; (b) while the sample is moving
through a section of the channel (i) cycling the temperature of the
sample in order to achieve PCR, (ii) illuminating the sample with
excitation light, and (iii) using a fiber optic probe to capture
fluorescent light emitted from the sample; and (c) measuring the
intensity of the fluorescent light.
[0029] In some embodiments, the fiber optic probe includes: (i) an
optical fiber having a proximal end and a distal end and (ii) a
probe head connected to and positioned adjacent to the distal end
of the optical fiber, the probe head having a field of view and
being positioned such that at least a portion of the section of the
channel is within the field of view. The probe head may be
positioned directly above the channel and may be positioned no more
than about 10 millimeters from the top of the channel.
[0030] In some embodiments, the method further includes moving the
fiber optic probe along at least the section of the channel while
using the fiber optic probe to capture the fluorescent light. The
speed at which the fiber optic probe is moved along the section of
the channel may be greater than the speed at which the sample moves
through the channel (in some embodiments it may be at least 10
times greater).
[0031] In some embodiments, the method further includes using a
second fiber optic probe to capture fluorescent light emitted from
the sample while the sample is moving through the section of the
channel. In some embodiments, all the while the sample is moving
through the section of the channel, the first fiber optic probe and
the second fiber optic probe are fixed in position relative to the
channel.
[0032] The above and other embodiments of the present invention are
described below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The accompanying drawings, which are incorporated herein and
form part of the specification, illustrate various embodiments of
the present invention. In the drawings, like reference numbers
indicate identical or functionally similar elements.
[0034] FIG. 1 depicts a prior art apparatus to capture an image of
a fluorescent signal.
[0035] FIG. 2 is a block diagram illustrating a system according to
some embodiments of the invention.
[0036] FIG. 3 is a diagram of a close-up side view of a possible
probe head assembly.
[0037] FIG. 4 illustrates an embodiment of the present invention
wherein excitation light and fluorescence both travel along the
same optical fiber.
[0038] FIG. 5 illustrates an embodiment of the present invention
wherein excitation light and fluorescence both travel along the
different optical fibers in a bundle of optical fibers.
[0039] FIG. 6 illustrates an embodiment of the present invention
wherein the excitation light is directed on to the sample without
going through an optical fiber.
[0040] FIG. 7A depicts a cross-sectional view of a version of the
present invention utilizing a single probe.
[0041] FIG. 7B depicts a cross-sectional view of a version of the
present invention utilizing multiple probes.
[0042] FIGS. 8A-8D illustrate a few of many possible trajectories
probes may take to scan an area of interest.
[0043] FIG. 9 depicts a bundle of optical fibers.
[0044] FIG. 10 depicts a positioning system coupled to a probe.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0045] As used herein, the words "a" and "an" mean "one or
more."
[0046] Aspects of the present invention provide a system for
detecting fluorescence emitted from a microfluidic device using at
least one fiber optic probe.
[0047] FIG. 2 illustrates a functional block diagram of a system
200 according to some embodiments of the invention. As illustrated
in FIG. 2, system 200 may include a microfluidic device 202.
Microfluidic device 202 may be a microfluidic chip. Microfluidic
device 202 may include one or more microfluidic channels 204. In
the example shown, device 202 includes two microfluidic channels,
channel 204a and channel 204b. Although only two channels are shown
in the exemplary embodiment, it is contemplated that device 202 may
have fewer than two or more than two channels. For example, in some
embodiments, device 202 includes eight channels 204.
[0048] Device 202 may include two DNA processing zones, a DNA
amplification zone 231 (a.k.a., PCR zone 231) and a DNA melting
zone 232. A DNA sample traveling through the PCR zone 231 may
undergo PCR, and a DNA sample passing through melt zone 232 may
undergo high resolution thermal melting. As illustrated in FIG. 2,
PCR zone 231 includes a first portion of channels 204 and melt zone
232 includes a second portion of channels 204, which is down stream
from the first portion.
[0049] In order to achieve PCR for a DNA sample flowing through the
PCR zone 231, the temperature of the sample must be cycled, as is
well known in the art. Accordingly, in some embodiments, system 200
includes a temperature control system 220. The temperature control
system 220 may include a temperature sensor, a heater/cooler, and a
temperature controller. In some embodiments, a temperature control
system 220 is interfaced with main controller 230 so that main
controller 230 can control the temperature of the samples flowing
through the PCR zone and the melting zone.
[0050] To monitor the PCR process and the thermal melting process
that occur in PCR zone 231 and melt zone 232, respectively, system
200 may include an imaging system 218. Imaging system 218 may
include an excitation source 253, a detector 250, a controller 251,
and an image storage unit 252.
[0051] Further features of system 200 are described in U.S. patent
application Ser. No. 11/770,869, which is incorporated herein by
this reference in its entirety.
[0052] Referring now to FIG. 3, an embodiment of imaging system 218
is illustrated. As shown in FIG. 3, imaging system 218 may include
a fiber optic probe 301 that includes a probe head 302 connected to
an optical fiber 306, which directs fluorescent light to a light
sensor or detector array 250. Suitable detectors would include, but
not be limited to: photomultiplier tubes; micro-channel plate
detectors; photoconductors; photodiodes (include avalanche
photodiodes); and detector arras including CCD and CMOS detector
arrays. Fixed and/or tunable wavelength filters 308 discriminate
against unwanted wavelengths such as scattered excitation light. In
addition, the fluorescence may be dispersed spectrally onto a
plurality of detectors by using devices such as diffraction
gratings, prisms, or multilayer dielectric wavelength filters.
[0053] Excitation light may be directed onto the microfluidic
device 202 in the same location where the probe head 302 is set to
collect emitted light. The excitation light may comprise light of
multiple wavelengths and may be generated by a variety of light
sources. In addition, excitation light may be directed onto the
microfluidic device 202 in a variety of ways. In one embodiment,
the excitation light source 253 is coupled to the same fiber 306
used to carry captured fluorescence with coupling optics 316. This
embodiment may use, for example, a dichromatic filter 314 to direct
excitation light through coupling optics 316 and into the optical
fiber 306 on substantially the same path as the fluorescence, but
in the opposite direction.
[0054] Referring now to FIG. 4, a diagram of a close-up side view
of one possible probe head 302 is shown. In general, when light is
emitted from the microfluidic channel 204a of microfluidic chip
202, it follows light path 412 and through probe head 302 is
collected into the optical fiber 306. It would be understood by one
of ordinary skill in the art that optical fiber 306 may comprise a
single optical fiber or, as shown in FIGS. 9 & 10, a bundle of
optical fibers.
[0055] As shown in FIG. 4, probe head may include a light
collecting element 408 connected to the distal end 402 of each
optical fiber 306. Light collecting element 408 may comprise one or
more of a high-index spherical lens, gradient index lens, a Fresnel
lens, a micro-lens system, a lensed fiber, or any combination
thereof. Probe head 302 may further comprise a spacer 410
positioned between the end 402 of the fiber 306 and light
collecting element 408. Preferably, probe head 302 is integrally
connected to the optical fiber 306.
[0056] Probe head 302 is designed to capture a significant fraction
of the light emitted from within a channel of the microfluidic
device 202. By positioning the probe head 302 close to the outer
surface of the device, it is possible to achieve reasonably high
collection efficiency with a relatively small diameter collecting
element 408. In one embodiment, desirable collection efficiencies
can be achieved by positioning the probe head about 20 millimeters,
and preferably about 10 millimeters, from the top of a channel of
the microfluidic device 202. Of course, other distances between the
probe head and the top of the channel may be used as well.
[0057] A scanner 490 can be connected to the probe head to scan the
probe head across an area of interest. Scanner 490 may include a
positioner (e.g., the MX80 positioner available from Parker
Hannifin Corporation of PA ("Parker")) for positioning probe head
302, a stepping drive (e.g., the E-AC Microstepping Drive available
from Parker) for driving the positioner, and a controller (e.g.,
the 6K4 controller available from Parker) for controlling the
stepping drive.
[0058] Referring now to FIG. 5, another embodiment of imaging
system 218 is illustrated. In the embodiment shown, the excitation
light can be carried by at least one separate optical fiber. As
shown in FIG. 5, light from the excitation source or sources 253 is
directed through the coupling optics 516e to excitation optical
fiber 506e. Similarly, fluorescence from the probe head 302 is
directed from fluorescence optical fiber 506f through the coupling
optics 516f to filters 308 and the detector or detectors 250.
Fluorescence optical fiber 506f and excitation optical fiber 506e
can be bundled together to form optical bundle 506b.
[0059] Referring now to FIG. 6, another embodiment of imaging
system 218 is illustrated. In the embodiment shown in FIG. 6, the
excitation light is not carried by a fiber, but is directed into
the micro-channel through free space. As shown in FIG. 6, light
from the excitation light source or sources 253 is directed on to
the microfluidic chip 202 using mirror 620. Mirror 620 may be
movable so as to be capable of directing the excitation light on to
any desired point on the microfluidic chip 202.
[0060] Referring now to FIGS. 7A and 7B, a comparison of a single
fiber optic probe to a multiple fiber optic probe configuration is
shown. A single probe 702 may be connected to a scanner that scans
the probe over an area of interest from different locations in
series. The single probe 702 can be scanned in one or two
dimensions across the face of the microfluidic chip 710.
[0061] Alternatively, in the embodiment shown in FIG. 7B, a
plurality of fiber optic probes 704 may be fixed with respect to
the microfludic chip 710, and each collects fluorescence signal
data from one location. As an alternative to being fixed, the
plurality of probe heads 704 could be configured so that they can
be scanned over an area of interest and each probe head 704 can be
used to gather image data from a section of the total area of
interest.
[0062] An advantage of using multiple probes at the same time is
that use of multiple probes creates some degree of parallelism and
could be used to decrease the time required to collect the desired
image data. This is especially true if the probe diameter is
comparable to, or smaller than the required spatial resolution in a
particular direction.
[0063] Depending on a combination of parameters (e.g. probe head
size, required spatial resolution, required signal acquisition
durations, etc.), a number of possible acquisition sequences and
scanning trajectories are possible. FIGS. 8A-8D illustrate a few of
the possible scanning trajectories. As shown in FIG. 8A, a 2-D
scanner trajectory 802 could be used with a single probe. In this
trajectory, the probe is moved over an area of interest in two
dimensions. As shown in FIG. 8B, by using more probes (e.g. five)
an "almost" 1-D scanner trajectory 804 may be adopted wherein
several are scanned in one direction and then back again. The
trajectory of FIG. 8C uses even more probes. A 1-D scanner
trajectory 806 can be used wherein the several probes are only
moved across the area of interest in one direction. FIG. 8D shows
another trajectory option: a fixed array 808 of probes.
[0064] Referring now to FIGS. 9 and 10, another embodiment of probe
head 302 is illustrated. Probe head 302 can comprise perimeter
probe heads 902 and one or more inner probe heads 904. Each probe
head 902 and 904 can be connected to a different optical fiber 306
or 1002 (as shown in FIG. 10). Optical fibers 306 can direct
excitation light through perimeter probe heads 902 and optical
fiber 1002 can direct fluorescence collected by inner probe head to
a light detector. Each perimeter probe head 904 may emit the same
frequency of excitation light or different frequencies. It would be
understood by those of ordinary skill in the art that different
configurations are also possible. For instance, perimeter probe
heads could be connected to a detector to detect fluorescence or
different detectors and inner probe head could emit excitation
light. Alternatively, some combination of inner and perimeter probe
heads could emit excitation light and some combination of inner and
perimeter probe heads could detect fluorescence.
[0065] While various embodiments/variations of the present
invention have been described above, it should be understood that
they have been presented by way of example only, and not
limitation. Thus, the breadth and scope of the present invention
should not be limited by any of the above-described exemplary
embodiments. Further, unless stated, none of the above embodiments
are mutually exclusive. Thus, the present invention may include any
combinations and/or integrations of the features of the various
embodiments.
[0066] Additionally, while the processes described above and
illustrated in the drawings are shown as a sequence of steps, this
was done solely for the sake of illustration. Accordingly, it is
contemplated that some steps may be added, some steps may be
omitted, and the order of the steps may be re-arranged.
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